This invention relates to methods of diagnosing flavivirus infection.
There are approximately 70 members of the flavivirus genus, a group of viruses that are antigenically very closely related, and distinguishable only by a specific neutralization test method (Calisher et al., J. Gen. Virol. 70:37-43, 1989). Approximately half of the 70 flaviviruses pose current or potential threats to public health. For example, Japanese encephalitis virus is a significant public health problem, involving millions of persons at risk in the Far East. Dengue virus, causing an estimated annual incidence of 100 million cases of primary dengue fever and over 450,000 cases of dengue hemorrhagic fever worldwide, has emerged as the single most important arthropod-transmitted human disease. In addition, West Nile virus, which causes febrile illness, occasionally complicated by acute encephalitis, is widely distributed throughout Africa, the Middle East, the former Soviet Union, and parts of Europe, and has recently become a concern in the eastern United States.
Other flaviviruses continue to cause endemic diseases of variable nature and have the potential to emerge into new areas as a result of changes in climate, vector populations, and environmental disturbances caused by human activity. These flaviviruses include, for example, St. Louis encephalitis virus, which causes sporadic, but serious, acute disease in the midwest, southeast, and western United States; Murray Valley encephalitis virus, which causes endemic nervous system disease in Australia; and Tick-borne encephalitis virus, which is distributed throughout the former Soviet Union and eastern Europe, where its Ixodes tick vector is prevalent and responsible for a serious form of encephalitis in those regions.
Hepatitis C virus (HCV) is another member of the flavivirus family, with a genome organization and a replication strategy that are similar, but not identical, to those of the flaviviruses mentioned above. HCV is transmitted mostly by parenteral exposure and congenital infection, is associated with chronic hepatitis that can progress to cirrhosis and hepatocellular carcinoma, and is a leading cause of liver disease requiring orthotopic transplantation in the United States.
Because of the impact of flaviviruses on worldwide public health, it is important that there be diagnostic tests that can be used to specifically and easily detect flavivirus infection in samples, in a variety of hospital or laboratory settings. Several types of antibody-based tests have been used to detect flavivirus infections, including enzyme-linked immunoassays (ELISAs), complement-fixation assays, hemagglutination-inhibition assays (HAIs), and neutralization assays. The first three assays listed are advantageous because they can employ inactivated antigens. This is important because work with live flaviviruses generally must be carried out in Biosafety Level 3 (BSL3) laboratories, which most pubic health and diagnostic facilities do not have. However, these assays are not without disadvantages. For example, these assays measure antibody binding at epitopes that are cross-reactive among different flaviviruses, making it very difficult to determine which particular flavivirus may be present in a sample. This is especially important in settings in which multiple flaviviruses co-exist. For example, in the United States, both St. Louis encephalitis and West Nile virus co-exist. Indeed, the original (1999) epidemic of West Nile encephalitis in New York was initially misdiagnosed at St. Louis encephalitis, because antibodies in patient""s blood cross-reacted with St. Louis encephalitis in ELISAs. In Australia, Murray Valley encephalitis, Japanese encephalitis, West Nile, and Kokobera viruses co-circulate and can cause diagnostic confusion. Similar considerations apply throughout Asia and Latin America, where multiple flaviviruses infect humans and animals.
The fourth assay listed above, the neutralization assay, overcomes this shortcoming of the other assays, in that it measures only neutralization epitopes, which vary between different flaviviruses. In fact, the 70 species within the flavivirus genus are distinguished based on differences in the neutralization test. Unfortunately, however, neutralization tests, which provide the specificity required to identify a particular flavivirus, cannot be used in most settings, because utilization of live virus, and thus BSL3 facilities, are required for these tests. Thus, the field would benefit from the development of a test that has the specificity of a neutralization test, but that would not require the use of a BSL3 facility.
Flaviviruses are members of a family of small, enveloped positive-strand RNA viruses. Flavivirus proteins are produced by translation of a single, long open reading frame to generate a polyprotein, and a complex series of post-translational proteolytic cleavages of the polyprotein by a combination of host and viral-proteases, to generate mature viral proteins (Amberg et al., J. Virol. 73:8083-8094, 1999; Rice, xe2x80x9cFlaviviridae,xe2x80x9d In Virology, Fields (ed.), Raven-Lippincott, New York, 1995, Volume I, p. 937). The virus structural proteins are arranged in the order C-prM-E, where xe2x80x9cCxe2x80x9d is capsid, xe2x80x9cprMxe2x80x9d is a precursor of the viral envelope-bound M protein, and xe2x80x9cExe2x80x9d is the envelope protein. These proteins are present in the N-terminal region of the polyprotein, while the non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are located in the C-terminal region of the polyprotein. The amino termini of prM, E, NS1, and NS4B are generated by host signalase cleavage within the lumen of the endoplasmic reticulum (ER), while most cleavages within the non-structural region are mediated by a viral protease complex known as NS2B-NS3 (Rice, supra). In addition, the NS2B-NS3 protease complex is responsible for mediating cleavages at the C terminus of both the C protein and the NS4A protein (Amberg et al., supra).
The invention provides methods of specifically detecting antibody against a predetermined virus that is present in a biological sample. The methods involve: (a) providing a chimeric virus having the following characteristics: (i) the chimeric virus includes nucleic acid sequences derived from two different viruses, one of which is the predetermined virus, the other being a flavivirus that is used as the chimera backbone; (ii) the chimeric virus is capable of replicating; (iii) the chimeric virus is substantially neutralized by antibodies to the predetermined virus and is not neutralized, or is neutralized to a lesser degree, by antibodies to other viruses, including the flavivirus used as the chimera backbone; (iv) the chimeric virus is attenuated compared to the predetermined virus; and (v) the chimeric virus may be safely manipulated in the laboratory at a Biosafety level that is lower than that required for the predetermined virus; (b) contacting the biological sample with the chimeric virus under neutralizing conditions; and (c) determining the presence or amount of infectious virus remaining following step (b) as an inverse measure of antibody against the predetermined virus that is present in the biological sample.
Step (c) of this method can involve, for example, inoculating a mammal with the sample-contacted chimeric virus, and then determining virus-induced illness or mortality in the mammal as a measure of non-neutralized infectious chimeric virus. Alternatively, step (c) can involve inoculating a cell culture with the sample-contacted chimeric virus, and then determining cytopathic effects, absence of metabolic activity, absence of uptake of vital dyes, or plaque formation as a measure of non-neutralized infectious chimeric virus.
The chimeric virus can include a backbone (e.g., yellow fever 17D virus) in which one or more structural genes (e.g., prM-E genes) have been replaced by the corresponding structural genes of the predetermined virus. The predetermined virus can be a flavivirus, for example, a mosquito-borne virus selected from the group consisting of Japanese encephalitis, Dengue (serotype 1, 2, 3, or 4), Yellow fever, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, and Ilheus viruses; a tick-borne flavivirus selected from the group consisting of Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses. All of these viruses are members of the family Flaviviridae. The principal underlying the use of chimeric viruses for diagnostic tests is the complete replacement of the envelope genes of the vector backbone (e.g., yellow fever 17D), so that the only remaining neutralization epitopes are those of the predetermined virus.
Genes containing neutralization epitopes or neutralization epitopes themselves from other members of the family Flaviviridae, but outside the genus Flavivirus (e.g., viruses from the Hepacivirus genus (e.g., a Hepatitis C virus) or Pestivirus genus (e.g., Bovine viral diarrhea virus), or from viruses outside the family Flaviviridae can also be used as donors for construction of chimeric viruses, which in turn may be used for detection of antibodies by neutralization test. For example, genes or epitopes from dangerous pathogens such as Lassa virus, Ebola virus, or Marburg virus could be inserted into a suitable backbone from an attenuated virus (e.g., yellow fever 17D), yielding a chimeric virus that may be safely manipulated in the laboratory for detection of neutralizing antibodies. In these cases, part of the envelope genes of the vector backbone (e.g., yellow fever 17D), would remain in the chimeric virus. For use in diagnostic tests, the vector virus (e.g., yellow fever 17D) would be used as a control to ensure specificity of the reaction to the gene or epitope of interest.
The invention also provides kits that can be used to carry out the methods described herein. The kits can contain a chimeric virus (see above) that includes structural proteins (e.g., prM and E proteins) of a predetermined virus, the presence of which in a sample can be tested for by use of the kit, or can include a nucleic acid molecule corresponding to the genome of such a chimeric virus. The kit can also include, for example, neutralizing antibodies against the predetermined virus, additional controls, buffers that can be used in the assays, instructions for carrying out the diagnostic methods described herein, vessels in which various steps of the method are carried out (e.g., tubes and/or trays). The kits of the invention are described in further detail below.
The invention provides several advantages. For example, as is noted above, previous assays used for diagnosing flavivirus infection employed either inactive viral antigens, and thus lacked the specificity required to distinguish between closely related flaviviruses, or employed live virus in neutralization assays and, thus, while providing high levels of specificity, required the use of BSL3 facilities. The present invention, involving the use of attenuated viruses, enables the use of the more sensitive and specific neutralization assay, but does not require the use of BSL3 facilities. The specificity of the methods of the invention enables medical professionals to pursue a course of treatment that is specific for the particular disease to be treated in a subject. The methods of the invention also enable public health and environmental professionals to track which particular viruses may be present in animal (e.g., mammal or bird) populations. In addition, these methods can be carried out in facilities having a lower Biosafety level (e.g., BSL2) than would be required for carrying out similar assays using wild type virus (e.g., BSL3 facilities).